The spliceosome is a dynamic macromolecular machine that catalyzes the excision of non-coding introns from pre-messenger RNAs (pre-mRNA) to form mature messages (mRNA), a process called pre- mRNA splicing. The ~3 MDa complex, composed of RNAs and proteins, assembles in a series of regulated steps from four small nuclear ribonucleoprotein subunits (snRNPs; U1, U2, U5, U4/U6) and numerous non- snRNP splicing factors. While the spliceosome is a fundamental cellular machine, its complex and dynamic nature has made obtaining structural information challenging. There are no structures of multi-snRNP (small nuclear ribonucleic particle) complexes at resolutions better than 30. The molecular organization of RNA and proteins within the spliceosome at any stage of the splicing reaction is not known and the global conformational changes that occur during the transitions from a pre- to post-splicing complex have not been characterized. The goal of this proposal is to define the conformational changes required for the transition from a pre- to post- activated spliceosome. In Aim 1 we will compare the structures of pre-activated, activated, and post-activated spliceosomes to map the global conformational changes required for pre-mRNA splicing. In Aim 2 we will define the molecular organization and map proximal protein-protein interaction networks of pre-activated, activated, and post-activated spliceosomes. In Aim 3 we will use structure/function studies to determine the role of the SF3 complex during catalytic activation and how this function is altered in Myelodysplastic Syndromes (MDS) and Chronic Lymphocytic Leukemia (CLL) patients that have mutations in this sub-complex. This work will provide direct insight into the conformational changes required for activation and define spliceosome organization during the transitions from a pre- to post-activated complex. Completion of these aims will significantly advance our understanding of the structure and organization of the spliceosome under conditions of both health and disease.